Abstract: The main purpose of this study was to express the neopullulanase gene of Paenibacillus polymyxa in Escherichia coli and to produce neopullulanase by high density fermentation. The neopullulanase gene npu was amplified from Paenibacillus polymyxa genomic DNA, the coding region contained 2106 bp in length and encoded 515 amino acids. The similarity of npu gene sequence with other neopullulanase genes of Paenibacillus polymyxa was 99%. The npu gene was successfully ligated with the plasmid PMD18T to construct the recombinant vector PMD18T-npu vecter, which was subsequently transformed into Escherichia coli BL21.After induced by methanol, npu gene was expressd in E. coli cells, and the enzyme could be secreted to the outside of E.coli cells. The recombinant strain BL21 PGEX 4t-1-npu had good genetic stability, and the total enzyme activity of the strain remained at 714 U after 10 generations of continuous culture.The enzyme activity of the purified enzyme solution remained at 5427 U after 6 months' storage at 25℃, and remained at 5390.5 U after 12 months' storage at 4℃. This is the first report of molecular characterization of an pullulanase from a strain of the genus paenibacillus. Npu is an attractive potential candidate for applications in starch processing and detergent industries due to its ability to effectively hydrolyze α-(1, 6) -linked branches in starch.